malERA: An updated research agenda for basic science and...

COLLECTION REVIEW

malERA: An updated research agenda for

basic science and enabling technologies in

malaria elimination and eradication

The malERA Refresh Consultative Panel on Basic Science and Enabling Technologies*¶

¶Membership of the malERA Refresh Consultative Panel on Basic Science and Enabling Tech-

nologies is listed in the Acknowledgments.� [email protected], [email protected], [email protected]

Abstract

Basic science holds enormous power for revealing the biological mechanisms of disease and,

in turn, paving the way toward new, effective interventions. Recognizing this power, the 2011

Research Agenda for Malaria Eradication included key priorities in fundamental research that,

if attained, could help accelerate progress toward disease elimination and eradication. The

Malaria Eradication Research Agenda (malERA) Consultative Panel on Basic Science and

Enabling Technologies reviewed the progress, continuing challenges, and major opportunities

for future research. The recommendations come from a literature of published and unpub-

lished materials and the deliberations of the malERA Refresh Consultative Panel. These

areas span multiple aspects of the Plasmodium life cycle in both the human host and the

Anopheles vector and include critical, unanswered questions about parasite transmission,

human infection in the liver, asexual-stage biology, and malaria persistence. We believe an

integrated approach encompassing human immunology, parasitology, and entomology, and

harnessing new and emerging biomedical technologies offers the best path toward address-

ing these questions and, ultimately, lowering the worldwide burden of malaria.

Summary points

• The recent development of multiple in vitro systems for studying malaria biology has

helped deepen our understanding of the disease. Nevertheless, research remains ham-

pered by a lack of in vitro models that can probe key aspects of malaria (e.g., gametocyte

development in Plasmodium vivax, fertilization, ookinete biology, parasite–midgut

interactions, human hepatocyte infection) and generate biological materials (i.e., infec-

tious sporozoites) for laboratory study. Developing the necessary cell lines and other in

vitro culture tools to propel these studies represent important areas for future research.

• With the emergence of widespread insecticide resistance in mosquito populations, there

is a strong need to bring basic research in mosquito biology back into the malaria

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002451 November 30, 2017 1 / 29

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OPENACCESS

Citation: The malERA Refresh Consultative Panel

on Basic Science and Enabling Technologies

(2017) malERA: An updated research agenda for

basic science and enabling technologies in malaria

elimination and eradication. PLoS Med 14(11):

e1002451. https://doi.org/10.1371/journal.

pmed.1002451

Published: November 30, 2017

Copyright: © 2017 The malERA Consultative Panel

on Basic Science and Enabling Technologies. This

is an open access article distributed under the

terms of the Creative Commons Attribution

License, which permits unrestricted use,

distribution, and reproduction in any medium,

provided the original author and source are

credited.

Funding: MESA received a grant from the Bill &

Melinda Gates Foundation (OPP1034591). The

funders had no role in study design, data collection

and analysis, decision to publish, or preparation of

the manuscript.

Competing interests: I have read the journal’s

policy and the authors of this manuscript have the

following competing interests: EAW, JHA, JB, SNB,

FC, BSC, MTD, AAJ, AKL, ML, IM, RR, DFW, and

SKV receive funding from the Bill & Melinda Gates

Foundation. EAW additionally receives funding

from the Medicines for Malaria Venture, NIH, and

serves on the Scientific Advisory Board of the Tres

Cantos Open Lab Foundation.

https://doi.org/10.1371/journal.pmed.1002451

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eradication agenda to strengthen current insecticide-based control campaigns and gen-

erate alternate vector control strategies.

• Driven by the development and accessibility of large-scale research tools and technolo-

gies, the scientific community can systematically tackle key questions in malaria, such as

the following. What are the genes that contribute to antimalarial drug resistance

(thereby defining the full parasite “resistome”)? What are the functions of key Plasmo-dium genes (providing much-needed annotation of key Plasmodium genes)? What are

the genes and gene mutations that drive resistance in mosquito populations?

• Continued exploration of the potential of enabling technologies is needed. Important

areas of future research include the use of gene-drive strategies and other gene-manipu-

lation technologies; metabolomics-based approaches for biomarker discovery; structural

vaccinology, novel technology platforms, and the use of novel adjuvants to improve vac-

cine design; and high-throughput approaches to facilitate drug discovery and screening.

Background

Since the first agenda for malaria eradication was published in 2011 [1], there have been many

significant developments in basic science, including an enhanced understanding of parasite

biology (both gametocyte and liver stages) as well as mosquito biology (Table 1). Some of these

advances could not have been predicted 5 years ago, such as the use of mouse models engrafted

with human liver to advance the biology of liver-stage parasites (including the quiescent P.

vivax hypnozoite stage) and the development of powerful genome-editing capabilities based

on clustered regularly interspaced short palindromic repeats/associated protein-9 nuclease

(CRISPR/Cas9) technology. In contrast, little progress has been achieved in several key

research areas that were previously prioritized and, as such, they remain important stumbling

blocks on the road to eradication.

We focus here on these and other crucial areas—deficiencies in basic science research and

the lack of enabling technologies—that currently limit our progress towards malaria elimina-

tion and eradication. Importantly, this analysis highlights specific aspects of the Plasmodiumlife cycle in both the human host and the Anopheles vector. Our integrated approach aims to

combine research efforts and expertise across human immunology, parasitology, and entomol-

ogy to introduce powerful new ideas and technologies from other fields, provide a multifaceted

view of disease biology, and accelerate progress toward eradication.

Methods

The findings presented in this paper result from an extensive literature review of published

and unpublished materials and the deliberations of the 2015 Malaria Eradication Research

Agenda (malERA) Refresh Consultative Panel on Basic Science and Enabling Technologies.

Electronic databases were systematically searched for published literature between January 1,

2010, and July 2, 2016, without language limitations. Panelists were invited to recommend

additional literature and additional ongoing research projects. A 2-day workshop was held

with the majority of the panel members, including field researchers, specialists from basic sci-

ence, malaria genomics and epigenomics, regenerative medicine, and National Institutes of

Health representatives. The panel broke into 6 breakout sessions to identify the problems that


Abbreviations: ap2, activator protein 2; CRISPR/

Cas9, clustered regularly interspaced short

palindromic repeats/associated protein-9 nuclease;

dsRNA, double-stranded ribonucleic acid;

ENCODE, Encyclopedia Of DNA Elements Project;

GWAS, genome-wide association study; IRS,

indoor residual spraying; LLIN, long-lasting

insecticide-treated net; malERA, Malaria

Eradication Research Agenda; MMV, Medicines for

Malaria Venture; TetR, tetracycline repressor.

¶Membership of the malERA Refresh Consultative

Panel on Basic Science and Enabling Technologies

is listed in the Acknowledgments.


need to be solved in asexual blood stages, liver stage and mosquito, mosquito, P. vivax,

population genetics and resistance, and transmission. The panel discussed what research is

needed to address these problems and considered 6 crosscutting themes in CRISPR

Table 1. A listing of the important research areas highlighted in malERA 2011, the progress made since then, and the remaining areas that require

additional research.

Research Area Accomplishments in Past 5 years References Remaining Gaps

Transmission Biology

(Gametocytes to

Mosquito)

Improved understanding of transcriptional and epigenetic

control of sexual development

[2–6] Limited work on P. vivax gametocytes due to

lack of in vitro culture system

Drug screens targeting transmission stages [7–11]

Improved understanding of mosquito host-seeking behavior

and olfaction biology

[12–16]

Improved understanding of mosquito–parasite interactions [17–20]

Anopheles midgut cell line model for in vitro ookinete

production and invasion

[21–25]

Infection Biology

(Mosquito to Liver)

Humanized mouse model for entire life cycle of Plasmodium,

including P. vivax hypnozoites and liver stages

[26, 27] Methods to increase sporozoite availability

In vitro models for Plasmodium liver stages [28–30]

Genetic crosses in mouse model [31]

Primate models for P. cynomolgi [32]

Controlled human malaria infections with sporozoites and

blood-stage parasites

[33–40],

reviewed in [41]

Biology of Blood-stage

Parasites

Improved production of continuous culture conditions,

including identification of host cell environments necessary

to support P. vivax invasion in culture and proof-of-principle

that human hematopoietic stem cells can be immortalized,

expanded, and differentiated into reticulocytes

[42–55] No in vitro culture system for P. vivax asexual

stages has been developed

Poor functional annotation of genes

P. knowlesi in vitro culture adaptation [56, 57]

Identification and spread of mutations associated with

artemisinin resistance

[58–64]

reviewed in

[65–67]

Comparison of mitochondrial and lipid metabolism of P.

falciparum in sexual and asexual blood stages

[68, 69]

Persistence of Parasites

and Mosquitoes

P. vivax hypnozoites cultured in vitro [26, 28] Biomarkers for asymptomatic hosts

Ecology and migration rates of vector species

Long-term behavioral resistance studiesMosquito dry season estivation and long-distance migration

observed in sub-Sahelian populations

[70]

Mechanisms of insecticide resistance identified [71–73]

Additional Technological

Developments

Mosquito genomic resources to identify population

substructure and allow comparative genomic studies

[74–77] Coordinated efforts to generate knockout or

knockdown libraries to understand gene

function, especially in human parasitesGenome-editing systems (CRISPR/Cas9, Zinc-finger

nuclease), posttranslational protein knockdown systems

(DD tag, Riboswitch), conditional genome deletion systems

(Cre-LoxP, FLP-frt, diCre), conditional gene expression

system (TetR-aptamer)

[78–86]

Proofs-of-principle for population suppression and

population modification/replacement of Anopheles using

gene drives

[87–92]

Colonization of important mosquito vector species [93]

New techniques to improve antigen design and clinical

evaluation of vaccine candidates

[94–100]

Improved resolution in intravital imaging [101, 102]

Abbreviations: Cre-LoxP, genetic recombination system involving the Cre (Causes recombination) protein and loxP (locus of X-over P); CRISPR/Cas9,

clustered regularly interspaced short palindromic repeats/associated protein-9 nuclease; diCre, dimerizable Cre recombinase; DD, destabilization domain;

FLP-frt, Flipase used to recombine two frt domains; malERA, Malaria Eradication Research Agenda; TetR, tetracycline repressor.

https://doi.org/10.1371/journal.pmed.1002451.t001


https://doi.org/10.1371/journal.pmed.1002451.t001


technologies, immunology and malaria vaccines, genomics tools for malaria, metabolism and

malaria, structural biology, and diagnostics for malaria. Each group fed back to plenary ses-

sion, where further robust discussions and input occurred. This helped refine the opportuni-

ties and gap areas in which research is needed. The final findings were arrived at with inputs

from all panelists and several iterations of the manuscript.

Advances, challenges, and opportunities in transmission biology

Gametocytes

Plasmodium transmission begins with the development of sexual forms of the parasite (known

as gametocytes) in an infected human host and their subsequent transfer to an anopheline

mosquito following a blood meal (Fig 1). This stage represents a key bottleneck in the parasite

Fig 1. Schematic depicting the human and mosquito life cycles of Plasmodium, highlighting critical questions at specific points within the life

cycle.

https://doi.org/10.1371/journal.pmed.1002451.g001


https://doi.org/10.1371/journal.pmed.1002451.g001


life cycle and thus is an attractive opportunity for disrupting disease transmission. As shown

in Box 1, in the past 5 years significant and exciting progress has been made in understanding

Box 1. Opportunities for the next 5 years

1. Functional genomics

• Identification of regulatory sequences within the parasite genome, similar to the

human Encyclopedia Of DNA Elements (ENCODE) project,

• Genome wide annotation of gene function in human parasites to identify sets of

genes involved in discrete cellular processes, including drug resistance,

• Improved scalability of CRISPR/Cas9 technology in asexual parasites to allow for

both pooled, genome-wide approaches (large scale) and single cell transformation

(microscale),

• Greater collaboration between researchers to avoid overlapping gene annotation

efforts.

2. Advances in mosquito biology

• Generation of a mosquito consortium to evaluate promising gene drive-based strate-

gies for efficacy at scale and/or over time and share knockout and/or transgenic

strains,

• Greater understanding of mosquito behavior and ecology,

• Colonization of important vector species,

• Development of in vitro mosquito infection models.

3. New vaccine approaches

• Improved adjuvants and identification of new targets, including better structures for

existing (and new) targets to improve structural approaches,

• Development of novel approaches with the potential to generate sterilizing immu-

nity (i.e., cognate antigens),

• Coordinated functional annotation of asexual-stage parasites to enable prioritization

of functional vaccine antigens,

• Greater access to samples and data from both human challenge studies and patient

samples demonstrating natural immunity,

• Application of gene-editing technologies to systematically understand the function

of hypothetical genes.

4. Biomarkers and diagnostics

• Indicators of transmissible gametocytes,

• Markers of liver-stage infection, in particular, hypnozoites,



gametocyte development, including insights into the transcriptional and epigenetic control of

sexual differentiation and evidence for bone marrow sequestration [2–6, 103]. In the case of P.

falciparum, newly available in vitro systems for gametocyte maturation have been used in

small molecule screening, antibody reagent development, and transcriptional and metabolo-

mics analyses [7–11].

In contrast, the mechanisms of P. vivax gametocyte development remain largely unknown.

Gametocyte biology within this species is quite distinct—development takes just 2 to 3 days

and unfolds prior to any clinical symptom. P. vivax gametocytes appear susceptible to existing

antimalarial drugs that are not effective against P. falciparum gametocyte stages [104–106].

Progress in this area has been hampered by the absence of a comparable in vitro culture system

for asexual P. vivax parasites, which is an urgent priority, as it would enable the generation of

gametocytes for laboratory study, mosquito infections, and sporozoite production.

Another major area for discovery is the elucidation of the biological determinants of game-

tocyte transmissibility, especially in areas of low endemicity. Does the success of transmission

depend on gametocyte quantity and/or quality? Are there mosquito-specific factors that

actively recruit gametocytes to the biting site or do gametocytes preferentially sequester near

the skin? What factors and mechanisms enable male and female gametes to find one another

in the mosquito midgut? Biomarkers for transmission competency could enable a broader

understanding of the heterogeneity in natural infections.

Mosquito biology and host seeking

Transmission success also depends upon the interactions of the mosquito vector with both its

human host and ingested parasites. Since 2011, there have been major advances in understand-

ing the biology of olfaction and host-seeking behavior in mosquitoes via a combination of

behavioral assays, electrophysiology, and functional genomic approaches [12–16]. High-

throughput screens have identified new classes of attractants and repellents that are currently

being tested in mosquito traps and spatial repellent trials ([107–110], also see MESA Track at

http://www.malariaeradication.org/mesa-track). Moving forward, the identification of

• Markers/assays to identify asymptomatic carriers,

• Identification of metabolic signatures of different stages of the life cycle.

5. Greater understanding of resistance to antimalarials and insecticides

• Identification of genes and pathways (i.e., the “resistome”) involved in resistance,

• Development of alternatives to insecticides,

• Use evolutionary approaches to prevent resistance.

6. Greater accessibility to P. vivax gametocytes

• Development of a P. vivax in vitro culture system (e.g., ookinetes to validate trans-

mission-blocking vaccine targets),

• Greater collaboration between groups to improve access to existing sporozoite

sources. This would be coupled with advances in cryopreservation to improve access

to sporozoites globally.


http://www.malariaeradication.org/mesa-track


oviposition cues and the role of olfaction and taste in larval stages could facilitate the develop-

ment of additional tools for vector control. Comparative genomic analysis of odorant receptor

pathways that differ between anthropophilic and zoophilic species will help to elucidate the

molecular basis of host-seeking behavior. Recent studies have shown that the composition of

the human skin microbiota influences host attractiveness to mosquitoes [111] and identified

volatile substances produced by parasites in human hosts thought to preferentially attract mos-

quitoes to infected individuals [112]. Nevertheless, gaps remain in our knowledge regarding

the potential for gametocyte-seeking behavior by the mosquito and parasite-induced changes

to the human host that may influence mosquito behavior to enhance biting and transmission.

Parasite development in the mosquito

Fertilized zygotes develop into the motile ookinete, which in turn crosses the midgut wall.

Major advances have been made in understanding midgut invasion and early mosquito anti-

Plasmodium immune responses that target the ookinete stage. Several parasite genes that inter-

act with the vector to enable its invasion of epithelial cells have been identified [17–19], and

new insights have emerged regarding the role of epithelial responses to invasion and the corre-

sponding epithelial interactions with the complement-like system to limit ookinete survival

[113–117]. There is increasing evidence that the oocyst stage is also a target of innate immunity

in the mosquito [118, 119]. Genome-wide association study (GWAS) mapping of Anophelespopulations displaying different vector competence has identified mosquito genes that influence

parasite development [120]. This list of potential targets to disrupt malaria transmission could

be extended through functional screens using double-stranded ribonucleic acid (dsRNA)-medi-

ated gene silencing in mosquitoes and synthetic approaches such as single-chain antibodies to

block P. falciparum from infecting salivary glands.

A particular challenge for developing new interventions is the lack of culture systems to study

fertilization, ookinete biology, and parasite–midgut interactions in human malaria parasites. Plas-modium species of rodents and birds have provided rapid proof-of-principle for new transmis-

sion-blocking strategies [121–123] and will likely continue to be critical for revealing the basic

biology of sexual and mosquito stages. The development of mosquito midgut-derived cell lines

(or organoids) supporting the in vitro culture of ookinetes and oocyst of human malaria parasites

would enable high-throughput transcriptomic and metabolomic studies as well as high-resolution

functional analysis of the parasite’s surface proteins and their interactions with mosquito cells.

These assays could also be used to validate transmission-blocking drugs and vaccines.

Advances, challenges, and opportunities in infection biology

The past 5 years have seen rapid progress in understanding the biology of Plasmodium infection

in the human liver. Increased availability of primary human hepatocytes has allowed the devel-

opment of multiple in vitro platforms, all tailored toward the concept of a miniaturized experi-

mental liver model [28, 29, 124]. Importantly, these innovations have allowed the liver stages of

infection to be fully recapitulated outside the human host for the first time [26, 125]. They have

also spurred the development of reagents to explore the biology of sporozoite infectivity and

liver stage development and provided the first glimpse of the P. vivax hypnozoite [26, 28].

In parallel, the development of humanized mouse models of P. vivax and P. falciparuminfection have opened up the potential for surrogate in vivo models of human liver infection

[26] and allowed the first genetic crosses of parasites (P. falciparum) outside of a primate [31].

Studies in primates continue to play an important role; the P. cynomolgi monkey model of

liver infection is the only in vivo relapse model of the P. vivax hypnozoite [30, 32, 126]. Com-

bined with controlled human malaria infections [34, 35, 38, 127, 128] and in vitro models,



these tools have highlighted key differences in the biology of different parasite species (specifi-

cally, P. vivax and P. falciparum) and paved the way for understanding the cellular biology of

liver infection and the immune response and for performing high-throughput drug candidate

screening.

To facilitate efforts aimed at eradication, we have identified a number of transformative

actions in the field of infection biology. A transformative innovation would be the in vitro cul-

tivation of large numbers of infectious P. falciparum and P. vivax sporozoites, bypassing the

mosquito vector. This would not only facilitate basic research but also contribute to whole-par-

asite vaccine development. Alternatively, advances in the preservation of sporozoite viability

and infectivity after mosquito dissection and/or the engineering of mosquitoes to produce spo-

rozoites at high levels would increase the availability and distribution of infectious material for

research purposes.

Improved liver-stage cell lines could also have a transformative effect on the pace of novel

drug and vaccine development, especially for P. vivax [28–30]. Cell lines provide readily avail-

able, immortal, and genetically identical cells, allowing researchers to reliably obtain the same

sensitivity measurements for each compound or antibody. This development could enable

high-throughput drug screening for discovery of liver stage-specific compounds targeting

either parasite functions [129] or human targets necessary for parasite development. More-

over, the availability of robust and inexpensive in vitro hepatocyte infection models for P.

vivax and P. falciparum may allow the development of better in vitro assays for antibody-

dependent inhibition of invasion (akin to virus neutralization assays) and cell-mediated killing

of infected cells. This could allow the discovery of human monoclonal antibodies with broadly

neutralizing activity, whose cognate antigens could then be used to create vaccines that give

sterilizing immunity. Recent advances in proteomics and mass spectrometry may also support

the identification of biomarkers for exoerythrocytic stages that are relevant in vivo.

Advances, challenges, and opportunities in asexual-stage biology

Defining the parasite “resistome”

Notable advances in asexual biology over the past 5 years include improvements in functional

genomics, such as more robust RNA sequencing methods [130–132], a deeper understanding

of transcription factors such as activator protein 2 (ap2) transcription factors [133] or alterna-

tive RNA splicing [134], and whole genome sequencing and genotyping of both field isolates

and evolved cultures (see Table 1). Due to its rapidly decreasing cost and increasing accuracy,

sequencing has accelerated our understanding of the mechanisms and modes of action of cur-

rent and new antimalarials through drug-resistant parasite selection in vitro (reviewed in

[135]) as well as population genetics of the parasite in vivo [62, 136]. Although numerous

studies have described using in vitro evolution and whole genome analysis to both find targets

of new antimalarial compounds and identify genes conferring resistance [62, 137, 138], in

most cases, only a handful of genes were identified. Now that single cell sequencing is becom-

ing a reality [139], we are in a position to identify every gene (and potentially allele) that con-

tributes to drug resistance, thus defining the parasite “resistome.” The complete genetic basis

of parasite drug resistance should provide better molecular markers of whether parasites have

acquired resistance to drugs that may be used in elimination campaigns, informing drug or

drug combination selections (See malERA Refresh paper on resistance [140]).

Systematic characterization of the asexual-stage parasite

The systematic knockout of genes in P. berghei has led to numerous advances in our under-

standing of fundamental asexual biology [141, 142], including the P. berghei identification of



essential genes and pathways [143–146], greater understanding of merozoite invasion and

egress [147–150], discovery of the parasite’s export machinery [145, 151, 152], and revealing

how the red cell cytoplasm and membrane are remodelled [153, 154]. Such studies point to the

critical nature of these processes and have opened the possibility of targeting them with drugs

or vaccines.

Yet, major gaps remain in our knowledge of gene function in P. falciparum and, to an even

greater extent, in other species (including P. vivax, P. ovale, and P. malariae) in which genetic

diversity is also relatively uncharacterized. Although in many cases, genomic variants can be

readily identified in sequencing data, poor annotations for predicted genes in the P. falciparumgenome continue to slow progress. For example, we know little about the cellular function of

the pfkelch13 gene, a major contributor to artemisinin resistance ([62, 155, 156], reviewed in

[67]). Given that it is more efficient and inexpensive for the community to work together to

functionally annotate the P. falciparum genome systematically rather than in a 1-researcher-

1-gene fashion, coordinated large-scale projects with a focus on the easily accessible P. falcipa-rum asexual blood stage should be considered. Such systematic data would also help in the

interpretation of whole genome sequencing data from drug- or vaccine-resistant parasites.

Desirable genomic annotations include the location of key transcription factor binding sites,

transcriptional start and stops sites [157], epigenetic chromatin modifications, and the cellular

localization of encoded proteins. These consortium-acquired data are critical to predict whether

genetic variants discovered through genome sequencing of model organisms and humans are

indeed functional and could also help prioritize antigens for vaccine development. In addition,

if better in vitro culture systems can be developed for P. vivax (see “Advances, challenges, and

opportunities in transmission biology”), these systematic approaches could be extended to this

important species. A potential model for such a consortium-based effort is the human

ENCODE project, which has identified functional elements in the human genome [158].

Using metabolomics to identify biomarkers and develop diagnostics

There have been major advances in the use of modern mass spectrometry-based methods for

identifying and profiling metabolites from parasite-infected cells [159–161] as well as determin-

ing the mode of action of drugs through the metabolic perturbations of exposed parasites [162–

165]. Two key areas in which metabolomics-based approaches have yet to make a significant

impact are biomarkers and diagnostics. Given the difficulty and cost associated with identifying

infected individuals (particularly those who are asymptomatic—see malERA Refresh paper on

reservoir and transmission [166]), the development of effective metabolomic biomarkers with

significant correlation to infection would represent a critical advance. Furthermore, to deter-

mine host markers of infection, field samples across a broad range of infectivities, including

asymptomatic carriers, should be studied using metabolomic methods. Such analyses should

also aim to span all Plasmodium parasite species as well, particularly P. vivax.

The question of persistence: Where do parasites—And

mosquitoes—Hide?

In the drive towards elimination and eradication, a key question is how and where malaria

infection persists in both humans and mosquitoes, both in individuals as well as populations.

Recent genomic studies indicate that parasites may also persist in an additional zoonotic reser-

voir in nonhuman primates [167–169], although how this contributes to disease transmission

in humans is currently unclear.

Persistence of malaria occurs in 2 modalities—asymptomatic carriers and latent liver stages.

The asymptomatic carriers represent a significant threat to the reintroduction of malaria; thus,



the identification of such carriers requires a heightened level of awareness and detection. The

absence of symptoms in an individual may reflect the presence of disease-prevention host

responses in the absence of sterilizing immunity, thereby allowing persistent parasitemia or

the sequestration of parasites in sites (e.g., the liver or bone marrow) in which they are “hid-

den” from the immune system. Understanding the relative contributions of both human

immune responses and parasite biology will be essential to maximize the efficacy of antimalar-

ial interventions, particularly vaccines.

Parasite persistence in the liver is a major hurdle for elimination efforts, particularly for P.

vivax, because of its rapid development of gametocytes in humans, enabling transmission

before the onset of clinical symptoms. Insights have emerged from studies of nonhuman pri-

mate models and humanized mouse models [26] in which parasite forms resembling hypno-

zoites demonstrated some biologic activity. These findings imply that sensitive technologies,

such as proteomics and metabolomics, may identify markers likely secreted at these stages.

Such markers would require field validation but ultimately could be incorporated into point-

of-care diagnostics, eliminating the need for primaquine or tafenoquine in mass drug adminis-

tration campaigns and informing epidemiological studies of the load of hypnozoite infection

in endemic regions.

The transmission of Plasmodium infections with low or submicroscopic levels of circulating

gametocytes suggests the possibility of nonrandom sequestration of gametocytes at sites in

peripheral skin that are accessible to mosquitoes. P. falciparum gametocytes have recently been

found to have an extended maturation period in the bone marrow [103, 170]. A clear implica-

tion of this observation, however, is that gametocytes detected in the peripheral circulation

may not accurately reflect overall or infectious gametocyte levels and that more sensitive assays

are needed to identify potential sources of transmission.

Mosquito vector persistence

The aspects of vector biology that enable malaria persistence remain to be investigated and will

be critical not only for informing and targeting current elimination and eradication strategies

but also for the development and successful deployment of novel vector-based interventions.

Recent data suggest that, in Africa, both mosquito estivation (dry season diapause) and long-

distance migration contribute to the persistence of sub-Sahelian mosquito populations follow-

ing a dry season, but in a species-specific manner [70]. New genomic resources have facilitated

the understanding of fine-scale mosquito population structures [77, 171] suggesting large and

stable populations [74–76]. The contribution of the observed genomic patterns to population

persistence is unclear at this point, and a better understanding of the life history, ecology, and

migration rates of vectors that result in the observed genomic patterns between populations is

needed. Similar studies in non-African mosquito populations are needed.

Mosquitoes also persist through physiological resistance to insecticides (see malERA

Refresh paper on resistance [140]), either through target site mutations, increased expression

of detoxifying enzymes, or cuticular thickening. Genomic markers associated with resistance

continue to be identified, yet together they do not adequately explain all the variation in insec-

ticide resistance phenotypes observed in natural populations, and their relative functional

impact in the field remains poorly understood.

Mosquito persistence may also occur due to heritable changes in behavior selected for by

control interventions, so-called behavioral resistance. Recent work has captured mosquito

interactions with bednets using mosquito-tracking cameras [172] and could be extended to

other interventions (e.g., traps, sprays, repellents). Consistent longitudinal studies are also

needed to track changes in mosquito biting behavior (e.g., outdoor versus indoor, evening



versus night) after the use of interventions and to discriminate these changes from variation in

species frequencies at specific sites. Subsequent genomic analyses could then reveal if there is a

genetic component to these modified behaviors.

Technology and its application to malaria biology

Fundamental technologies: Genomics and transcriptomics

Whole genome sequencing has already had a major impact on multiple areas of parasite and

vector research. It has transformed our understanding of parasite biology and drug resistance

(see “Advances, challenges, and opportunities in asexual-stage biology”). In addition, it has

been widely used to study the population genetics of mosquito species in the field [74–76,

173], and the genomes of 19 Anopheles species spanning 3 subgenera and including major and

minor malaria vectors from diverse geographical locations have now been sequenced [77,

171]. These genomic resources have improved our understanding of the patterns of gene flow

within and among mosquito populations. These “big data” resources available to the research

community allow for powerful comparative functional and evolutionary analyses that will help

elucidate the common basis of vector competence and identify effective vector control targets

across multiple species. Recent work using these datasets has identified a reproductive trait

with consequences for vectoral capacity that has evolved within the Anopheles genus and pres-

ents new potential targets to induce sterility in field populations [174–176]. Additional targets

may be identified as our understanding of the biological coordination of simultaneous egg

development and parasite transmission is improved. The declining cost of sequencing will

make such studies more feasible in the future, such that a mosquito resistome—similar to the

parasite resistome—may be compiled.

Further advances in genomic technology will enable a detailed analysis of natural popula-

tions of Plasmodium spp. at a worldwide scale. These include single cell technologies for

genome sequencing and transcriptomic analyses, genotyping, and whole genome sequencing

from dried blood spot samples. In addition, further comparative genomics [177] among all

Plasmodium species infecting humans as well as those infecting nonhuman primates should

identify key pathways in host switching. Genomic analysis of longitudinal samples will allow

for the identification of population structure changes associated with changing epidemiology

and emerging drug resistance. Coupled with gene-editing technologies, hypotheses generated

by comparative genomics can be functionally tested.

Technical advances in RNA sequencing now make it feasible to interrogate the dynamic

gene expression profiles of both the human host and the parasite during infection. This will

provide new insights into the host response during infection and the potential adaptation of

parasites during the infective process.

Gene-manipulation technologies: Genome editing and transgenics

Genome engineering tools, such as CRISPR/Cas9 systems (see glossary in the malERA Refresh

Introductory paper [178]), have transformed the ability to manipulate the genomes of P. falcip-arum, P. berghei (reviewed in [179]), and Anopheles and understand gene function. CRISPR/

Cas9-based genetic engineering of P. falciparum asexual blood stages has allowed for more

complex genetic modifications within the parasite; for example, the tetracycline repressor pro-

tein (TetR) aptamer system to control gene expression [84] utilized CRISPR/Cas9 as an initial

step to introduce the aptameric cassette. Beyond CRISPR/Cas9, however, there have been sev-

eral other successful gene-editing technologies (see Table 1).

With these powerful tools in place, we can now scale up the generation of conditional and/

or complete knockout parasite libraries containing every single gene in the genome. Such an



effort would greatly enhance our understanding of the biology of the parasite at all stages of

development, as well as identify the functions of many hypothetical genes.

Gene-manipulation technologies: Gene drives

Mirroring the advances in gene-editing capabilities in the parasite, Anopheline spp. genomes

can also now be engineered with unprecedented precision (see Table 1). Recent reports show

that CRISPR/Cas9 gene-editing tools can be used for the generation of gene-drive systems [91,

92] that manipulate genetic inheritance in mosquitoes to spread anti-Plasmodium transgenes

(population modification/replacement strategies) or lethality-inducing transgenes (population

suppression strategies) through natural mosquito populations. Mendelian inheritance predicts

50% of offspring will inherit a transgene carried on one of a parent’s chromosomes. Genetic

drive is the increased transmission of a genetic element to over 50% of offspring so that it

increases in frequency in each generation. A gene drive typically refers to an artificial trans-

gene that shows genetic drive by giving it the ability to trigger its own replication. A gene-drive

transgene is copied from one chromosome to its homologous chromosome within germ line

cells. With both chromosomes carrying a copy of the transgene (a homozygous germ line), all

sperm or eggs derived from these cells will also carry the transgene, and if copying occurs in all

germ cells, 100% of offspring will inherit the gene drive. This allows rapid spread of the gene

drive (and its anti-Plasmodium cargo) into the mosquito population. A valuable debate on the

safe use of gene drive systems has begun within the scientific community [180].

The feasibility of using gene drive strategies for mosquito control will need additional

research efforts in 3 key areas. First, an understanding of mosquito mating biology and the

determinants of male mating success and female mate choice will need to be developed. Colo-

nization is likely to impact the mating ability of species that exhibit such a complicated mating

behavior as swarming; mating competitiveness will be a key determinant of gene drive success.

Second, effective, “evolution proof” gene-drive systems should be generated to preempt the

selection of mosquitoes that are resistant to the drive mechanisms, which would otherwise

reduce the efficiency of the drive. Gene drives will need to be optimized by testing different

gene-drive architectures, especially if CRISPR/Cas9 mechanisms prove problematic. Third,

effective antimalarial genes will need to be evaluated in a reliable and reproducible manner;

many anti-Plasmodium factors have been identified and should be systematically tested in lab-

oratory conditions for their ability to block parasite development within the mosquito host.

Consideration should be given to the formation of a consortium to evaluate and prioritize

promising transgenic strategies and test these in multiple anopheline species and against a

number of Plasmodium isolates. This represents an opportunity to avoid duplication of work;

however, we would also argue for head-to-head comparison of transgenic strategies. Such a

consortium could centralize resources, particularly in developing transgenic mosquitoes (e.g.,

injection service, mail-order mutants) and potentially a mutant library, but, currently, the

space required for mosquito-line maintenance prevents this. As forward and reverse genetic

screens become more realistic, we should develop methods to cryopreserve mosquito lines or,

more realistically, store plasmids for injection to recreate lines as needed.

Cell- and tissue-based technologies

Since the discovery of malaria parasites by microscopy [181], imaging has played a central role

in malaria research. However, recent advances in imaging techniques have allowed visualiza-

tion of the parasite and its interactions with the mammalian host and insect vector at an

unprecedented level of resolution [101] [182] [102]. We can expect that imaging will reveal



other novel insights into the biology of human malaria parasites and play a major role in the

science of malaria eradication.

New technologies to support tool development: Biomarkers and novel

diagnostics

As our understanding of parasite biology advances—including insights into sequestration and

dormancy—the potential to leverage emerging technologies to support the discovery of bio-

markers of infection (see above) increases. Such insights into parasite biology are laying the

foundation for novel diagnostic approaches based on more sensitive techniques to detect para-

site byproducts (e.g., hemozoin) [183] or volatile substances [184]. When noninvasive, rapid,

and inexpensive, these diagnostic approaches are likely to facilitate the identification of

infected individuals who may be asymptomatic and/or functioning as reservoirs (see malERA

Refresh papers on Tools [185] and the Reservoir and Transmission [166]).

Exosomes are key new players implicated in intercellular communication without direct

cellular contact [186] and have a potential role as biomarkers [187]. The release of microparti-

cles is augmented in human malaria [188, 189], and exosomes containing parasite proteins

have been shown to be produced by infected cells [190] as well as by parasites [191, 192].

New technologies in vaccine development and leveraging existing

human volunteer sample datasets

Protective immunity requires that human hosts recognize and respond appropriately to para-

site-derived antigens and epitopes. Such immunity is complex, however, requiring both innate

and acquired responses and biological regulation of such responses as well as ensuring the

responses’ durability. Malaria parasites utilize a number of mechanisms to evade these immune

responses, which infected hosts must then overcome. In this context, there is a fundamental gap

in understanding the correlates of protective immunity in the human host that target exoeryth-

rocytic-stage parasites in both P. falciparum and P. vivax. Multiple new technologies are now

available to identify antigens and epitopes that are the targets of innate and acquired immune

responses. Examples include high-throughput genomic sequencing, transcriptomics, and prote-

omics. Structural vaccinology [193–195] has proven immensely powerful in viral vaccine devel-

opment through improved immunogen design and is now being applied to asexual blood stages

[94–97]. Near-atomic resolution cryo-electron microscopy is now being used to inform antigen

and drug target selection as well as the rational design of potent immunogens [196–198]. In

addition, new technology platforms and novel adjuvants are being incorporated into vaccines to

ensure appropriate immune responses are elicited. Approaches based on structural biology [98–

100] and genomic sequencing [199] are now being introduced into the clinical evaluation of

candidate malaria vaccines. These efforts provide an opportunity to further define the effective

targets as well as the nature of protective immune responses.

An effective P. vivax vaccine strategy also needs to contend with the challenge of relapse

infections. To prevent “relapse outbreaks,” antirelapse vaccines will need to be multistage and

multivalent, including components to suppress blood-stage parasites emerging from the dor-

mant liver stages as well as block transmission. There are relatively few P. vivax vaccine candi-

dates progressing currently through the global pipeline [200].

Controlled human challenge studies are potentially transformational in enabling our

understanding of the human immune response to malaria. Coupling controlled infections

with technical advances for interrogating human immune cells in real time can give us new

insights into both the temporal response and the contributions from innate and acquired

immunity. Additionally, deeper interrogation of the immune profile of naturally acquired



infections could also provide key insights. Providing access to them will require forethought in

preparing future proposals, particularly with respect to human subject approvals, repository

deposition, and community sharing. Harnessing available systems through existing networks

as well as ongoing clinical trials could provide the necessary reagents and access to human

samples.

Drug design and screening

The identification of potential targets through metabolomics and systems biology approaches

coupled with advances in structural biology is now facilitating the design of compounds likely

to interact with such targets. Moreover, high-throughput screening technologies are facilitat-

ing more rapid identification and prioritization of compounds for further investigation as

potential leads, though corresponding techniques in high-throughput synthesis and character-

ization of small molecules require further development. In a reverse approach, high-through-

put phenotypic screens are also enabling the selection of compounds whose structures can

subsequently be used to inform the identification of potential molecular interactions and met-

abolic pathways for further analysis as targets for pharmacologic intervention (reviewed in

[201]). It is important to note that because malaria primarily affects the developing world, the

opportunity for profit is reduced. Malaria, with the assistance of the community and funders

such as Medicines for Malaria Venture (MMV), has and will continue to function as a model

for open source drug discovery [202–204].

Technologies targeting mosquito-based interventions: Paratransgenesis

and genetically modified mosquitoes

Recent years have seen a focus toward the identification of microbial populations that can

block parasite development in the mosquito vector [205–208]. Genetic modification of these

bacterial populations (paratransgenesis) could be a key tool, particularly for the control of out-

door biting and resting mosquito populations that are not currently targeted by insecticide-

based strategies. Advances in Wolbachia bacteria experiments in Anopheles mosquitoes are

particularly promising. Wolbachia are intracellular endosymbiotic bacteria that, in some

insects, spread through populations by maternal transmission and cytoplasmic incompatibil-

ity. These endosymbionts were shown to block malaria parasite development in artificial set-

tings [209] and were negatively correlated with Plasmodium infections in natural A. coluzziipopulations from Burkina Faso [210, 211]. Two key research priorities are the development of

a method to transform Wolbachia to deliver effective antiplasmodial genes and understanding

the role of natural Wolbachia infections in malaria transmission dynamics.

In light of widespread resistance to currently used insecticides, the identification of alternative,

safe, active compounds that can extend the lifetime of long-lasting insecticide-treated nets (LLINs)

and indoor residual spraying (IRS) is imperative. The study of key pathways in mosquito repro-

duction, susceptibility to infection, blood feeding behavior, and longevity that can be effectively

targeted to reduce vectoral capacity is therefore a priority. For example, new sterilizing com-

pounds that interfere with key hormonal reproductive pathways, such as those regulated by juve-

nile hormone and 20-hydroxyecdysone, could be incorporated into mosquito nets to reduce

mosquito fertility, including insecticide-resistant mosquitoes that may survive exposure to the net.

A key issue in applying these novel strategies will be achieving effective colonization of

anopheline species, as the lack of mosquito colonies is preventing studies on the biology of

important malaria vectors. An important breakthrough has been the recent colonization of A.

darlingi, the most important American vector [93]. On the road to eradication, a deeper

understanding of the biology and behavior of these species will be essential.



Conclusions

As illustrated above, recent advances in basic science are providing deeper insights into the

biology of the parasite, the mosquito vector, and the human host as well as their interactions at

molecular, cellular, and organismic levels. Coupling these insights with recent technologies

that help pinpoint potential methods to intervene or disrupt essential interactions can spur the

use of novel tools to help eliminate and, ultimately, eradicate malaria.

Acknowledgments

The Malaria Eradication Research Agenda (malERA) Refresh Consultative Panel on Basic Sci-

ence and Enabling Technologies was chaired by Dyann F. Wirth, Harvard T.H. Chan School

of Public Health, United States of America, and cochaired by Elizabeth A. Winzeler, University

of California San Diego, United States of America, and B. Fenton (“Lee”) Hall, National Insti-

tute of Allergy and Infectious Diseases at the National Institutes of Health, United States of

America. The paper was written based on consultations during a malERA Refresh meeting

held in Boston, United States of America, December 7–9, 2015, plus additional literature

searches.

Members of the writing group who met the ICMJE criteria for authorship

Dyann F. Wirth (chair), Harvard T.H. Chan School of Public Health, United States of Amer-

ica; Elizabeth A. Winzeler (cochair), University of California San Diego, United States of Amer-

ica; B. Fenton (“Lee”) Hall, National Institute of Allergy and Infectious Diseases at the National

Institutes of Health, United States of America (cochair); Selina E. Bopp, Harvard T.H. Chan

School of Public Health, United States of America; Gregory LaMonte, University of California

San Diego, United States of America; N. Regina Rabinovich, Harvard T.H. School of Public

Health, United States of America and ISGlobal Barcelona Institute for Global Health, Spain; W.

Robert Shaw, Harvard T.H. Chan School of Public Health, United States of America.

Members of the panel

Dyann F. Wirth (chair), Harvard T.H. Chan School of Public Health, United States of

America; Elizabeth A. Winzeler (cochair), University of California San Diego, United States of

America; B. Fenton (“Lee”) Hall (cochair), National Institutes of Health, United States of

America; John H. Adams, University of South Florida, United States of America; Frederic

Ariey, Institut Cochin, Universite Paris Descartes Sorbonne, France; Carolina V. Barillas-

Mury, National Institutes of Health, United States of America; Jake Baum, Imperial College

London, United Kingdom; Sangeeta N. Bhatia, Massachusetts Institute of Technology, United

States of America; Oliver Billker, Wellcome Trust Sanger Institute, United Kingdom; Selina E.

Bopp, Harvard T.H. Chan School of Public Health, United States of America; Flaminia Catter-

uccia, Harvard T.H. Chan School of Public Health, United States of America; Alan F. Cow-

man, The Walter and Eliza Hall Institute of Medical Research, Australia; Chetan E. Chitnis,

Institut Pasteur, France; Brendan S. Crabb, Burnet Institute, Australia; Kirk W. Deitsch, Weill

Cornell Medical College, United States of America; Hernando A. Del Portillo, Barcelona Insti-

tute of Global Health, Spain; Abdoulaye A. Djimde, University of Bamako, Mali; Carlota

Dobaño, Barcelona Institute of Global Health, Spain; Patrick E. Duffy, National Institutes of

Health, United States of America; Manoj T. Duraisingh, Harvard T.H. Chan School of Public

Health, United States of America; Christian Happi, College of Natural Sciences and African

Center of Excellence for Genomics of Infectious Diseases, Redeemer’s University, Nigeria;

Anthony A. James, University of California, United States of America; Gregory LaMonte, Uni-

versity of California San Diego, United States of America; Amanda K. Lukens, Broad Institute

of MIT, United States of America; Manuel Llinas, Pennsylvania State University, United States

of America; Prashant Mali, University of California San Diego, United States of America;



Matthias Marti, University of Glasgow, Glasgow, United Kingdom; Jesus Martinez-Barnetche,

National Institute of Public Health, Mexico; Victoria McGovern, Burroughs Wellcome Fund,

United States of America; Maria M. Mota, Instituto de Medicina Molecular, Portugal; Ivo

Mueller, The Walter and Eliza Hall Institute of Medical Research, Australia and Institut Pas-

teur, France; Daouda Ndiaye, Cheikh Anta Diop University, Senegal; Daniel E. Neafsey, Broad

Institute of MIT and Harvard, United States of America; Francine Ntoumi, University of

Tubingen, Germany; Jetsumon Prachumsri, Mahidol University, Thailand; Pushkar Sharma,

National Institute of Immunology, India; W. Robert Shaw, Harvard T.H. Chan School of Pub-

lic Health, United States of America; Photini Sinnis, Johns Hopkins Bloomberg School of Pub-

lic Health, United States of America; Niraj H. Tolia, Washington University, United States of

America; Sarah K. Volkman, Harvard T.H. Chan School of Public Health, United States of

America; Edward A. Wenger, Institute for Disease Modeling, United States of America; Kim

Williamson, Uniformed Services University of the Health Sciences, United States of America.

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